Smooth muscly myosin light chain kinase incorporates two moles of phosphate per mole of myosin kinase when it is incubated with the catalytic subunit of cAMP-dependent protein kinase and ATP in the absence of bound calmodulin. In the presence of bound calmodulin one mole of phosphate is incorporated per mole of kinase. Two dimensional peptide mapping on thin layer cellulose has confirmed the presence of two different sites of phosphorylation, only one of which can be phosphorylated when calmodulin is bound to myosin kinase. These two sites appear to be present in both turkey gizzard myosin kinase and human platelet myosin kinase. Using brief tryptic digestion of native myosin kinase it was possible to first remove the two phosphorylated sites without loss of calmodulin dependence, and then the calmodulin binding site. The resulting fragment of myosin kinase was active in the absence of Ca++-calmodulin.